1. Field of the Invention
This invention relates generally to apparatus and methods for measuring assays and more specifically to a spectrophotometric apparatus and method for measuring assays prepared using standard wet chemistry procedures and conventional solid phase technology wherein the solid phase remains in the reaction cuvette during the measurement and is utilized as the light source for the measurement.
2. Statement of the Related Art
The use of spectrophotometric apparatus and methods to measure assays prepared using standard wet chemistry procedures and conventional solid phase, coated bead technology is well known. One typical procedure for performing and measuring an enzyme immunoassay (EIA), for example, involves coating a spherical plastic bead (the carrier or substrate) with an antibody or antigen which is the compliment of the antibody or antigen being tested for. The bead is then put through a series of sample exposure, reagent exposure, incubation, and washing steps that cause the surface of the bead to become coated with one or more layers (solid phases) of the antigen or antibody being tested for and the complimentary antigen or antibody. The antigen or antibody forming the outer layer is labelled with an enzyme such as horseradish peroxidase, for example. The coated bead is then exposed to a solution containing a chromophore such as OPD which reacts with the enzyme and colors the solution. The optical density of the coloration, i.e. the degree of optical development of the solution, is indicative of the concentration of the antigen or antibody being tested for in the original sample. A conventional spectrophotometer measures the assay by illuminating the solution with one or more beams of light having one or more selected wavelengths in the visible light range corresponding to spectral components of the colored solution. A detector measures the absorbance of the selected wavelengths by the solution. The measured absorbance values are then processed in a known manner to obtain the concentration of the antigen or antibody of interest.
Automated assay machines that spectrophotometrically measure batches of assays prepared using coated bead technology, such as the EIA just described, are known. Typically in such machines, a plurality of such assays are formed in a corresponding plurality of reaction wells or cuvettes of a molded plastic tray. The coated beads are removed from the cuvettes by a mechanical apparatus and placed in separate glass or plastic tubes before chromophore is added and measurements taken.
In order to improve the processing speed and throughput of such equipment, it is advantageous to be able to measure the assays without removing the coated beads from the reaction cuvettes. Attempts have been made in the past to spectrophotometrically measure an assay while retaining the solid phase in the reaction cuvette during the measurement. However, these attempts have required the use of specially designed assay components to avoid contact between the solid phase and the light beam during the measurement.
Cetus PCT Pat. No. 86/02168, for example, discloses an automated assay machine which spectrophotometrically measures the absorption of visible light by a reacted assay with the bead in the reaction well. The Cetus system requires specially designed reaction trays having individual wells with slanted, slotted bottom surfaces or separate sub-wells in order to maintain the bead outside the path of the vertical light beam which passes through the solution during the measurement.
Suovaniemi et al. U.S. Pat. No. 4,147,752 discloses a substrate for a solid phase assay that allows the assay to be measured without removing the substrate. The substrate is a specially designed form piece that must be positioned in the cuvette with a specific orientation. In one embodiment, the form piece is shaped as a ring to facilitate spectrophotometric measurement of the assay without contact between the light beam and the form piece.
In view of the above described need and the limitations of the prior art, it is an object of the present invention to provide a spectrophotometric apparatus that is capable of measuring assays prepared using conventional wet chemistry procedures and solid phase technology with the solid phase remaining in the reaction well during the measurement, and that is particularly suitable for use in automated assay equipment.
It is another object of the invention to provide such an apparatus that utilizes the solid phase itself as a light diffusing source to accomplish the measurement.
It is still another object of the invention to provide such an apparatus that is used advantageously with conventional reaction trays and coated beads.